A major goal of this project is to investigate the role of known oncogenes and genomic DNA from prostatic cancer cells in transforming normal human prostatic epithelial cells. The "immortalization" of a normal human prostatic epithelial cell line, NP-2s, has been accomplished. This line (Lechner et al., JNCI, 60: 797, 1978) was recovered from liquid nitrogen and cultured in serum-free medium (P4-8F) consisting of PFMR4 (Lechner et al., Methods in Cell Biology, Vol. 21B, pp. 195-225, 1980), without trace element concentrate, supplemented with selenite, 50 nM; calcium, 0.5 mM; epidermal growth factor, 0.5 ng/ml; insulin, 5.0 mug/ml; bovine pituitary extract, 0.5%; bovine serum albumin, 250 mug/ml; phosphoethanolamine, 0.5 muM; and cholera toxin, 0.1 nM. Overnight cultures of cells near the end of their lifespan were transfected with plasmid p-RSV-T consisting of the RSV-LTR promoter and the gene encoding the SV40 large T-antigen (Brash et al., Mol. Cell. Biol., May 1987). The treated cells formed rapidly-growing, multi-layered colonies within 2 weeks at a frequency of 1-2/10,000 cells at risk in 4 repeat experiments, whereas the untreated cells became quiescent and formed no colonies. Individual transfected colonies were isolated, expanded and tested for growth in suspension, tumorigenicity in nude mice, karyotype and response to growth factors. All 13 colonies tested are non-tumorigenic and remain anchorage-dependent. There was significant extension of the life span of NP-2 cells following transfection with constructs containing ras, v-myc or both. These non- tumorigenic lines will be used to investigate further steps toward neoplasia.